In recent years, glycomolecules have been gathering attention as the third chain-like biological molecule following nucleic acids (DNA) and proteins. The human body is one great big cellular society consisting of about 60 trillion cells, and all cell surfaces are covered with glycomolecules. For example, the ABO blood group system is determined by the difference of sugar chains on the cell surface.
Sugar chains have a function related to intercellular recognition and interaction, acting as a keystone in constituting the cellular society. Derangement in the cellular society will lead to e.g. cancer, chronic disease, infection, and aging. For example, it is known that structural change of sugar chains on the cell surface occurs when a cell becomes cancerous.
In addition, it is known that Vibrio cholerae or influenza virus etc. invade the cell and cause infection by recognizing and binding to a particular sugar chain.
The elucidation of such sugar chain functions leads to, e.g. the development of pharmaceutical agents or foods based on a new principle, and broad applications such as prevention of illnesses and contribution to therapies are expected.
Sugar chains have very complex structures compared to nucleic acid or protein structures due to diversities such as monosaccharide sequence, binding mode and site, chain length and branching mode, and general higher-order structure. Accordingly, the biological information derived from the structure of sugar chain is widely varied compared to that from nucleic acids or proteins. Although the importance of sugar chain research is acknowledged, the propulsion of research is in a delayed state compared to nucleic acids or proteins due to the complexity and diversity of the structure of sugar chain.
Many of the proteins present on the cell membrane surface or in the serum etc. have a sugar chain bound thereto. A molecule where a sugar chain is covalently bound to a protein is called a glycoprotein, and can be divided into two groups according to the difference in the binding mode of the sugar and the protein. One is the asparagine-linked sugar chain (N-glycosidic bond) where the amino group on the side chain of asparagine (Asn) is bound with the sugar chain. The other is the mucin-linked sugar chain (O-glycosidic bond) where the sugar chain is bound to the alcohol of serine (Ser) or threonine (Thr). All asparagine-linked sugar chains have a basic skeleton consisting of 5 sugar residues, and are classified into the subgroups of high mannose-type, complex-type, and hybrid-type according to the sugar residue type at the non-reducing terminal of the sugar chain bound. On the other hand, mucin-linked sugar chains are classified into four groups according to the difference in the basic skeleton (core).
Although proteins having a sugar chain have already been globally utilized as glycoprotein formulations, there were problems that these glycoproteins could only be obtained by methods mainly utilizing biotechnology, and that glycoproteins manufactured thereby were low in purity. Accordingly, a chemical synthesis method that efficiently affords the glycoprotein of interest in high purity was desired. Specifically, when synthesizing a glycopeptide having a non-naturally occurring amino acid, biological methods are not capable of direct production.
The solid phase synthesis method developed by R. B. Merrifield in 1963 is currently widely used as a peptide synthesis method. The solid phase synthesis method is a method where amino acid building blocks are joined on a solid phase called a resin and the peptide chain is elongated. When the peptide chain elongation is complete, the peptide chain is cleaved from the solid phase to afford the target object.
As an application of this, an amino acid bound to a sugar chain can be integrated upon peptide chain elongation to enable glycopeptide chain synthesis.
In the solid phase synthesis method, the amino group of the amino acids to be the building blocks is protected by e.g. a fluorenylmethoxycarbonyl (Fmoc) group, a tert-butoxycarbonyl (Boc) group, or a benzyloxycarbonyl (Cbz or Z).
In the solid phase synthesis method employing a Boc group, a super strong acid such as hydrogen fluoride is used for deprotecting the protecting group of the peptide side chain and cleaving out the peptide itself from the resin, and there was a problem that when a sugar chain is contained in a portion of the target object due to this hydrogen fluoride treatment, the sugar chain portion, especially the sialic acid present at the sugar chain non-reducing terminal is easily degraded. It was thus difficult to directly manufacture the glycoprotein of interest having a sialyl sugar chain with Boc solid phase synthesis method.
In the solid phase synthesis method employing an Fmoc group, the Fmoc group can be detached from the amino group of an amino acid under basic condition. On the other hand, since the Boc group can be deprotected from the amino group of an amino acid under acidic condition, Boc solid phase synthesis method is necessary when synthesizing a peptide or a glycopeptide employing a base-labile non-naturally occurring amino acid with solid phase synthesis method.
Non-naturally occurring amino acids are amino acids that do not configure proteins but some exists in nature, and can also be obtained by chemical synthesis. Non-naturally occurring amino acids have extremely high diversity of structure or flexibility of substituent selection. Improvement of in vivo stability, improvement of potency, improvement of absorption efficiency, improvement of distribution within tissue, and change of three-dimensional structure of peptide etc. can be expected by utilizing such a non-naturally occurring amino acid to synthesize a peptide, and non-naturally occurring amino acids are gathering attention as allowing designing of candidate substances for novel peptide medicines and functional materials.
As one solid phase synthesis method, there is also reported a method wherein the peptide produced when cleaving the peptide off from the resin is converted into a thioester form (e.g. Non-Patent Literature 1). Once a peptide in thioester form is obtained, it can be bound to other peptide chains by utilizing e.g. Native Chemical Ligation (NCL method) or Kinetically Controlled Ligation (KCL method), allowing a larger protein of interest to be manufactured (Patent Literature 1 and Non-Patent Literature 2).
The NCL method is a method of obtaining a larger peptide chain by linking a peptide fragment having Cys at the N-terminal amino acid and a peptide fragment having a thioester at the C-terminal. A glycoprotein can be synthesized by employing a glycosylated peptide fragment for this. Each fragment can be synthesized by for example the above solid phase synthesis method, and glycosylated fragments having uniform amino acid sequence and sugar chain structure can be obtained by binding a glycosylated asparagine having uniform sugar chains instead of an amino acid during synthesis (Patent Literature 2). In addition, the KCL method is a method of obtaining the glycoprotein of interest in relatively large amounts by utilizing the difference in reaction rate when both peptide fragments to be linked have a thioester at the C-terminal.
Accordingly, by performing the NCL or the KCL method with a glycosylated fragment, uniform glycoproteins that do not vary depending on the production lot and that can also be utilized as a pharmaceutical agent can be obtained.